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International audience; Untangling chromatin interactions The plant nucleus contains myriad subdomains that define and are defined by the composition of their chromatin. On a whole nucleus scale, this includes regions of active euchromatin and inactive heterochromatin, although the boundaries between these states are blurred. Different loci constantly enter and exit nuclear structures including the nucleolus and transient regulatory nuclear bodies. Chromatin is defined by its complex combinations of epigenetic modifications and variation in its constituent histone proteins. Here we preview articles that describe many of the mechanisms that regulate this dynamic chromatin.

Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.

Helianthemum squamatum is a specialist gypsophile, the only species of a recently diverged lineage in subgenus Helianthemum characterized by having the lowest chromosome number in the genus (n = 5). With the hypothesis of great genome reorganization in the lineage of H. squamatum, we (1) modelled the evolution of the chromosome number in the genus Helianthemum, (2) analysed the karyotype and the nuclear DNA content of H. squamatum and its sister species H. syriacum (n = 10) and (3) studied in detail the meiotic process of H. squamatum. Our analyses show that: (1) the rate of chromosome losses in the lineage that gave rise to H. squamatum is 100 times higher than in the genus as a whole; (2) compared to its sister species, H. squamatum has a more symmetric karyotype composed of longer metacentric chromosomes and retains c. 80% of its nuclear DNA content and (3) achiasmatic behaviour of chromosomes occurs during microsporogenesis despite full synapsis. Our results are in agreement with previous knowledge showing that reduced chromosome numbers in determinate lineages are found in short-lived species adapted to stressful environments, and we suggest that a combination of fewer chromosomes, a smaller genome, a shorter life cycle and the suppression of meiotic recombination can together contribute to the maintenance of those advantageous allelic combinations that makes H. squamatum a true gypsophile, enabling the individual plants to cope with the harshness imposed by dry gypsum soils.

Significance Cyclin-dependent kinases are the main drivers of the mitotic cell cycle. Here, we show that the activity of the main cell-cycle regulator in the model plant Arabidopsis, CDKA;1, also governs one of the most important processes in meiosis: the formation of meiotic cross-overs. We show that CDKA;1 activity especially affects the major class of meiotic cross-overs, known as class I cross-overs. We find that lowering kinase activity leads to a progressive loss of cross-overs, of which cross-overs near the chromosome ends are the last to disappear. Conversely, an increase of kinase activity increases the rate of cross-over formation., Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis. Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

Mitosis and meiosis in higher plants involve significant reconfiguration of the nuclear envelope and the proteins that interact with it. The dynamic series of events involves a range of interactions, movement, breakdown, and reformation of this complex system. Recently, progress has been made in identifying and characterizing the protein and membrane interactome that performs these complex tasks, including constituents of the nuclear envelope, the cytoskeleton, nucleoskeleton, and chromatin. This review will present the current understanding of these interactions and advances in knowledge of the processes for the breakdown and reformation of the nuclear envelope during cell divisions in plants.

Significance Meiotic recombination plays a fundamental role in shaping genetic diversity in eukaryotes. Extensive variation in crossover rate exists between populations and species. The identity of modifier loci and their roles in genome evolution remain incompletely understood. We explored natural variation in Arabidopsis crossover and identified SNI1 as the causal gene underlying a major modifier locus. To date, SNI1 had no known role in crossover. SNI1 is a component of the SMC5/6 complex that is closely related to cohesin and condensin. Arabidopsis sni1 and other SMC5/6 mutants show similar effects on the interference-independent crossover pathway. Hence, our findings demonstrate that the SMC5/6 complex, which is known for its role in DNA damage repair, is also important for control of meiotic crossover., The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1. The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

The nuclear envelope delineates the eukaryotic cell nucleus. The membrane system of the nuclear envelope consists of an outer nuclear membrane and an inner nuclear membrane separated by a perinuclear space. It serves as more than just a static barrier, since it regulates the communication between the nucleoplasm and the cytoplasm and provides the anchoring points where chromatin is attached. Fewer nuclear envelope proteins have been identified in plants in comparison with animals and yeasts. Here, we review the current state of knowledge of the nuclear envelope in plants, focusing on its role as a chromatin organizer and regulator of gene expression, as well as on the modifications that it undergoes to be efficiently disassembled and reassembled with each cell division. Advances in knowledge concerning the mitotic role of some nuclear envelope constituents are also presented. In addition, we summarize recent progress on the contribution of the nuclear envelope elements to telomere tethering and chromosome dynamics during the meiotic division in different plant species.

During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).

18 Pags.- 3 Figs. This article is available under the Creative Commons CC-BY-NC license and permits non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited., Genetic information in the cell nucleus controls organismal development and responses to the environment, and finally ensures its own transmission to the next generations. To achieve so many different tasks, the genetic information is associated with structural and regulatory proteins, which orchestrate nuclear functions in time and space. Furthermore, plant life strategies require chromatin plasticity to allow a rapid adaptation to abiotic and biotic stresses. Here, we summarize current knowledge on the organization of plant chromatin and dynamics of chromosomes during interphase and mitotic and meiotic cell divisions for model and crop plants differing as to genome size, ploidy, and amount of genomic resources available. The existing data indicate that chromatin changes accompany most (if not all) cellular processes and that there are both shared and unique themes in the chromatin structure and global chromosome dynamics among species. Ongoing efforts to understand the molecular mechanisms involved in chromatin organization and remodeling have, together with the latest genome editing tools, potential to unlock crop genomes for innovative breeding strategies and improvements of various traits., The work of all authors on this manuscript was stimulated and supported by the COST action CA16212 ‘Impact of Nuclear Domains On Gene Expression and Plant Traits’. AP was supported from the European Regional Development Fund (ERDF) project ‘Plants as a tool for sustainable global development’ (no. CZ.02.1.01/0.0/0.0/16_019/0000827), INTER-COST grant LTC18026 from the MEYS Czech Republic, and a Purkyně Fellowship from the Czech Academy of Sciences. CC was supported by the EU Horizon 2020 project TomGEM (no. 679796). IC was supported by the European Research Council Shuffle (ERC-Advanced to Professor R. Waugh; Project ID: 669182). KK was supported by a grant of the General Secretary for Research and Technology of Greece, Infrastructures support program [MIS5002803] ‘PlantUP’. SV was supported by Italian CNR Epigen Flagship Project. TK was supported by the US–Israel Binational Agricultural Research and Development Fund (US 4916-16). CM was supported from the ERDF project ‘Centre for Experimental Plant Biology’ (no. CZ.02.1.01/0.0/0.0/16_019/0000738), and grants from the MEYS Czech Republic (LTC18034) and the Czech Science Foundation (18-02448S). M-PV was supported by the project AGL2016-77211-R from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain. AM was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and ERDF (FEDER grant BIO2014-57011-R). MP acknowledges support from the Spanish Ministry of Economy and Competitiveness (MINECO) (AGL2015-67349-P) and Horizon 2020 Programme (Marie-Curie ITN MEICOM network FP7 ITN-765212).

Rubylation is a conserved regulatory pathway similar to ubiquitination and essential in the response to the plant hormone auxin. In Arabidopsis thaliana, AUXIN RESISTANT1 (AXR1) functions as the E1-ligase in the rubylation pathway. The gene AXR1-LIKE (AXL), generated by a relatively recent duplication event, can partially replace AXR1 in this pathway. We have analysed mutants deficient for both proteins and complementation lines (with the AXR1 promoter and either AXR1 or AXL coding sequences) to further study the extent of functional redundancy between both genes regarding two processes: meiosis and DNA repair. Here we report that whereas AXR1 is essential to ensure the obligatory chiasma, AXL seems to be dispensable during meiosis, although its absence slightly alters chiasma distribution. In addition, expression of key DNA repair and meiotic genes is altered when either AXR1 or AXL are absent. Furthermore, our results support a significant role for both genes in DNA repair that was not previously described. These findings highlight that AXR1 and AXL show a functional divergence in relation to their involvement in homologous recombination, exemplifying a duplicate retention model in which one copy tends to have more sub-functions than its paralog.

MicroRNAs (miRNAs) are a class of small (containing about 22 nucleotides) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level in plants and animals, being absent from unicellular organisms. They act on diverse key physiological and cellular processes, such as development and tissue differentiation, cell identity, cell cycle progression, and programmed cell death. They are also likely to be involved in a broad spectrum of human diseases. Particularly, this review examines and summarizes work characterizing the function of miRNAs in gametogenesis and fertility. Although numerous studies have elucidated the involvement of reproductive-specific small interfering RNAs (siRNAs) in regulating germ cell development and meiosis, less is known about the role of miRNAs in these processes. We focus on the study of hypomorphic and null alleles of genes encoding components of miRNA biogenesis in both plants (Arabidopsis thaliana) and mammals (Mus musculus). We compare the consequences of the presence of these mutations on male meiosis in both species.

During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).

Polyploid organisms provide additional opportunities to study meiosis in a more complex context since more than two potential homologous chromosomes are available. When the chromosome complement of a diploid individual is duplicated, each chromosome is accompanied by one identical and two homologous chromosomes within the same nucleus. In this situation, a competition in pairing/synapsis/chiasma formation between identical and homologous (but not necessarily identical) chromosomes can occur. Several studies have been conducted in different species to address whether there are preferences in crossover formation between identical rather than homologous chromosomes. In this study, multivalent and chiasma frequencies were cytologically analyzed in synthetic autotetraploids of Arabidopsis thaliana including the accessions Col, Ler, and the Col/Ler hybrid. Fluorescence in situ hybridization was conducted to identify each chromosome at metaphase I. The new Col and Ler tetraploids showed high multivalent frequencies, exceeding the theoretical 66.66% expected on a simple random end-pairing model, thus indicating that there are more than two autonomous synaptic sites per chromosome despite their small size. However, a significant excess of bivalent pairs was found in the Col/Ler hybrid, mainly due to the contribution of chromosomes 2 and 3. The mean chiasma frequencies of the three artificial autotetraploids were about twofold the corresponding mean cell chiasma frequencies of their diploid counterparts. The relative contribution of each chromosome to the total chiasma frequency was similar in the three genotypes, with the exception of a lower contribution of chromosome 3 in the hybrid. Preferences for chiasma formation between identical and homologous chromosomes were analyzed in Col/Ler 4x, taking advantage of the cytological differences between the accessions: variations in the size of the 45S rDNA region on the short arm of chromosome 2 and changes in the size and localization of the 5S rDNA region in chromosome 3. We observed a different behavior of chromosomes 2 and 3, i.e., random chiasma formation between identical and homologous chromosomes 2, and preferences for chiasma formation between homologous chromosomes 3. Hence, our results reveal the existence of chromosome-specific mechanisms responsible for these preferences.

The nuclear envelope (NE) is a dynamic boundary that allows the communication between nuclear and cytoplasmic components. It has essential roles in a variety of physiological processes including cell division. The linker of nucleoskeleton and cytoskeleton (LINC) complexes span the NE and are important during meiosis, the specialized cell division needed for sexual reproduction. During this division, the LINC complex proteins AtSUN1 and AtSUN2, located in the inner nuclear membrane (INM), are involved in tethering telomeres to the NE. This attachment promotes chromosome movements by the forces that are generated in the cytoplasmic face. In Arabidopsis, the double mutant Atsun1 Atsun2 exhibits a delayed prophase I meiotic progression, partial synapsis, and recombination defects that lead to the formation of unbalanced gametes and sterility. In meiocytes from these mutants, immunolabeling can be applied to analyze possible changes in the dynamics of different meiotic proteins. In addition, if the specific antibodies are available, this technique is an easy and useful tool to determine the spatial distribution of NE proteins.

Polyploid organisms provide additional opportunities to study meiosis in a more complex context since more than two potential homologous chromosomes are available. When the chromosome complement of a diploid individual is duplicated, each chromosome is accompanied by one identical and two homologous chromosomes within the same nucleus. In this situation, a competition in pairing/synapsis/chiasma formation between identical and homologous (but not necessarily identical) chromosomes can occur. Several studies have been conducted in different species to address whether there are preferences in crossover formation between identical rather than homologous chromosomes. In this study, multivalent and chiasma frequencies were cytologically analyzed in synthetic autotetraploids of Arabidopsis thaliana including the accessions Col, Ler, and the Col/Ler hybrid. Fluorescence in situ hybridization was conducted to identify each chromosome at metaphase I. The new Col and Ler tetraploids showed high multivalent frequencies, exceeding the theoretical 66.66% expected on a simple random end-pairing model, thus indicating that there are more than two autonomous synaptic sites per chromosome despite their small size. However, a significant excess of bivalent pairs was found in the Col/Ler hybrid, mainly due to the contribution of chromosomes 2 and 3. The mean chiasma frequencies of the three artificial autotetraploids were about twofold the corresponding mean cell chiasma frequencies of their diploid counterparts. The relative contribution of each chromosome to the total chiasma frequency was similar in the three genotypes, with the exception of a lower contribution of chromosome 3 in the hybrid. Preferences for chiasma formation between identical and homologous chromosomes were analyzed in Col/Ler 4x, taking advantage of the cytological differences between the accessions: variations in the size of the 45S rDNA region on the short arm of chromosome 2 and changes in the size and localization of the 5S rDNA region in chromosome 3. We observed a different behavior of chromosomes 2 and 3, i.e., random chiasma formation between identical and homologous chromosomes 2, and preferences for chiasma formation between homologous chromosomes 3. Hence, our results reveal the existence of chromosome-specific mechanisms responsible for these preferences.

The nuclear envelope (NE) is a dynamic boundary that allows the communication between nuclear and cytoplasmic components. It has essential roles in a variety of physiological processes including cell division. The linker of nucleoskeleton and cytoskeleton (LINC) complexes span the NE and are important during meiosis, the specialized cell division needed for sexual reproduction. During this division, the LINC complex proteins AtSUN1 and AtSUN2, located in the inner nuclear membrane (INM), are involved in tethering telomeres to the NE. This attachment promotes chromosome movements by the forces that are generated in the cytoplasmic face. In Arabidopsis, the double mutant Atsun1 Atsun2 exhibits a delayed prophase I meiotic progression, partial synapsis, and recombination defects that lead to the formation of unbalanced gametes and sterility. In meiocytes from these mutants, immunolabeling can be applied to analyze possible changes in the dynamics of different meiotic proteins. In addition, if the specific antibodies are available, this technique is an easy and useful tool to determine the spatial distribution of NE proteins.

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC-84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock-like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.

Plant ARGONAUTE (AGO) proteins regulate a wide range of cellular and developmental functions. Recent findings highlight their role during homologous recombination, a basic mechanism to repair double-strand DNA lesions (in somatic cells) and programmed DNA breaks (in meiocytes). This chapter contains an exhaustive description of procedures applied to analyze meiotic chromosome behavior (cytogenetic techniques) and DNA repair capacity (genotoxicity assays) in AGO-deficient Arabidopsis thaliana mutants.

Chromatin Assembly Factor 1 (CAF-1) is an evolutionary conserved heterotrimeric chaperone complex that facilitates the incorporation of histones H3 and H4 onto newly synthesized DNA. We demonstrate here that the mutant deficient for the large subunit of the complex, fas1-4, and in minor extent, the mutant deficient for the middle subunit, fas2-1, display chromosome abnormalities throughout Arabidopsis mitosis. Among them, we observed multicentromeric chromosomes at metaphase, and chromatid bridges and acentric fragments at anaphase-telophase. 45S rDNA and telomeric sequences were frequently involved in bridges and fragments. Gene expression analysis by real-time qPCR has revealed that several genes related to homologous recombination (HR) and alternative nonhomologous end-joining (aNHEJ) are overexpressed in fas1-4. These results concur with previous studies which have indicated that HR may be involved in the progressive loss of 45S rDNA and telomeres displayed by fas mutants. However, increased expression of PARP1, PARP2, and LIG6 in fas1-4, and the phenotype shown by the double mutant fas1-4 rad51 suggest that aNHEJ should also be responsible for the chromosomal aberrations observed. The activity of different DNA repair pathways in absence of CAF-1 is discussed.

In eukaryotic organisms, the correct regulation of sister chromatid cohesion, whereby sister chromatids are paired and held together, is essential for accurate segregation of the sister chromatids and homologous chromosomes into daughter cells during mitosis and meiosis, respectively. Sister chromatid cohesion requires a cohesin complex comprised of structural maintenance of chromosome adenosine triphosphatases and accessory proteins that regulate the association of the complex with chromosomes or that are involved in the establishment or release of cohesion. The cohesin complex also plays important roles in the repair of DNA double-strand breaks, regulation of gene expression and chromosome condensation. In this review, we summarize progress in understanding cohesion dynamics in plants, with the aim of uncovering differences at specific stages. We also highlight dissimilarities between plants and other eukaryotes with respect to the key players involved in the achievement of cohesion, pointing out areas that require further study.

MicroRNAs (miRNAs) are ~22-nt single-stranded noncoding RNAs with regulatory roles in a wide range of cellular functions by repressing eukaryotic gene expression at a post-transcriptional level. Here, we analyzed the effects on meiosis and fertility of hypomorphic or null alleles of the HYL1, HEN1, DCL1, HST and AGO1 genes, which encode miRNA-machinery components in Arabidopsis. Reduced pollen and megaspore mother cell number and fertility were shown by the mutants analyzed. These mutants also exhibited a relaxed chromatin conformation in male meiocytes at the first meiotic division, and increased chiasma frequency, which is likely to be due to increased levels of mRNAs from key genes involved in homologous recombination. The hen1-13 mutant was found to be hypersensitive to gamma irradiation, which mainly causes double-strand breaks susceptible to be repaired by homologous recombination. Our findings uncover a role for miRNA-machinery components in Arabidopsis meiosis, as well as in the repression of key genes required for homologous recombination. These genes seem to be indirect miRNA targets.

Plant ARGONAUTE (AGO) proteins regulate a wide range of cellular and developmental functions. Recent findings highlight their role during homologous recombination, a basic mechanism to repair double-strand DNA lesions (in somatic cells) and programmed DNA breaks (in meiocytes). This chapter contains an exhaustive description of procedures applied to analyze meiotic chromosome behavior (cytogenetic techniques) and DNA repair capacity (genotoxicity assays) in AGO-deficient Arabidopsis thaliana mutants.

Different histone modifications often modify DNA-histone interactions affecting both local and global structure of chromatin, thereby providing a vast potential for functional responses. Most studies have focused on the role of several modifications in gene transcription regulation, being scarce on other aspects of eukaryotic chromosome structure during cell division, mainly in meiosis. To solve this issue we have performed a cytological analysis to determine the chromosomal distribution of several histone H3 modifications throughout all phases of both mitosis and meiosis in different plant species. We have chosen Aegilops sp. and Secale cereale (monocots) and Arabidopsis thaliana (dicots) because they differ in their phylogenetic affiliation as well as in content and distribution of constitutive heterochromatin. In the species analyzed, the patterns of H3 acetylation and methylation were held constant through mitosis, including modifications associated with "open chromatin". Likewise, the immunolabeling patterns of H3 methylation remained invariable throughout meiosis in all cases. On the contrary, there was a total loss of acetylated H3 immunosignals on condensed chromosomes in both meiotic divisions, but only in monocot species. Regarding the phosphorylation of histone H3 at Ser10, present on condensed chromosomes, although we did not observe any difference in the dynamics, we found slight differences between the chromosomal distribution of this modification between Arabidopsis and cereals (Aegilops sp. and rye). Thus far, in plants chromosome condensation throughout cell division appears to be associated with a particular combination of H3 modifications. Moreover, the distribution and dynamics of these modifications seem to be species-specific and even differ between mitosis and meiosis in the same species.

The RNA-directed DNA methylation (RdDM) pathway is important for the transcriptional repression of transposable elements and for heterochromatin formation. Small RNAs are key players in this process by regulating both DNA and histone methylation. Taking into account that methylation underlies gene silencing and that there are genes with meiosis-specific expression profiles, we have wondered whether genes involved in RdDM could play a role during this specialized cell division. To address this issue, we have characterized meiosis progression in pollen mother cells from Arabidopsis thaliana mutant plants defective for several proteins related to RdDM. The most relevant results were obtained for ago4-1. In this mutant, meiocytes display a slight reduction in chiasma frequency, alterations in chromatin conformation around centromeric regions, lagging chromosomes at anaphase I, and defects in spindle organization. These abnormalities lead to the formation of polyads instead of tetrads at the end of meiosis, and might be responsible for the fertility defects observed in this mutant. Findings reported here highlight an involvement of AGO4 during meiosis by ensuring accurate chromosome segregation at anaphase I.

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

Information concerning natural variation either in chiasma frequency or in the genetic basis of any such variation is a valuable tool to characterize phenotypic traits and their genetic control. Here meiotic recombination frequencies are analysed in nine geographically and ecologically diverse accessions of Arabidopsis thaliana, and a comparative study was carried out incorporating previous data from another eight accessions. Chiasma frequencies, estimated by counting rod and ring bivalents at metaphase I, varied up to 22% among accessions. However, no differences were found among plants of the same accession. There was a relationship, which does not necessarily imply direct proportionality, between the size of the chromosomes and their mean chiasma frequency. Chiasma frequency and distribution between arms and among chromosomes were not consistent over accessions. These findings indicate the existence of genetic factors controlling meiotic recombination both throughout the whole genome and at the whole chromosome level. The reliability of chiasma scoring as an indicator of reciprocal recombination events is also discussed.

The present study is a sincere tribute from some of the cytogenetic laboratories of Madrid involved in the study of grasshopper meiosis, for the inestimable contribution of Professor Michael J.D. White to orthopteran cytogenetics, on the 100th anniversary of his birth. Here we review the knowledge accumulated over almost 90 y on the meiosis of the grasshopper Stethophyma grossum, especially on the male side, because of the very unusual meiotic characteristics shown by the spermatocytes, namely: whereas the three shortest bivalents achieve full synapsis and do not show chiasma localization and the single X chromosome remains asynapsed, the remaining eight bivalents show restricted synapsis and proximal chiasma localization. In addition, supernumerary segments and accessory chromosomes are present in some natural populations. Special attention is paid to the relationships among cohesin axis morphogenesis, initiation of recombination events and synapsis achievement.

Meiosis is a fundamental and evolutionarily conserved process that is central to the life cycles of all sexually reproducing eukaryotes. An understanding of this process is critical to furthering research on reproduction, fertility, genetics and breeding. Plants have been used extensively in cytogenetic studies of meiosis during the last century. Until recently, our knowledge of the molecular and functional aspects of meiosis has emerged from the study of non-plant model organisms, especially budding yeast. However, the emergence of Arabidopsis thaliana as the model organism for plant molecular biology and genetics has enabled significant progress in the characterisation of key genes and proteins controlling plant meiosis. The development of molecular and cytological techniques in Arabidopsis, besides allowing investigation of the more conserved aspects of meiosis, are also providing insights into features of this complex process which may vary between organisms.This review highlights an example of this recent progress by focussing on ASY1, a meiosis-specific Arabidopsis protein which shares some similarity with the N-terminus region of the yeast axial core-associated protein, HOP1, a component of a multiprotein complex which acts as a meiosis-specific barrier to sister-chromatid repair in budding yeast. In the absence of ASY1, synapsis is interrupted and chiasma formation is dramatically reduced. ASY1 protein is initially detected during early meiotic G2 as numerous foci distributed over the chromatin. As G2 progresses the signal appears to be increasingly continuous and is closely associated with the axial elements. State-of-the-art cytogenetic techniques have revealed that initiation of recombination is synchronised with the formation of the chromosome axis. Furthermore, in the context of the developing chromosome axes, ASY1 plays a crucial role in co-ordinating the activity of a key member of the homologous recombination machinery, AtDMC1.

Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants., Author Summary Meiosis is a special cell division common in all sexually reproducing organisms. It consists of two successive rounds of chromosome segregation, preceded by a single DNA replication event. Homologous recombination is a key process that occurs during the first meiotic division. It guarantees the association of the homologous chromosomes by chiasmata, the cytological manifestations of reciprocal interchanges (crossovers, COs). The formation of COs during meiosis is fine-tuned by several mechanisms. One of them, reported in some model organisms, is CO homeostasis, which ensures a consistent number of COs despite variability in early recombination events. Here we described the analysis of Atfas1-4, a mutant defective for the histone chaperone CAF-1 (Chromatin Assembly Factor 1), which has an increase in double-strand breaks (DSBs), without a corresponding increase in COs. Interestingly, Atfas1-4 has a higher gene conversion (GC) frequency. These results demonstrate that Arabidopsis meiocytes are able to maintain WT levels of COs even when DSBs numbers are increased. Furthermore, we provide evidence for a prominent role for AtDMC1 in establishing interhomolog interactions in Arabidopsis.

The allohexaploid Aegilops species (2n = 6x = 42), Ae. neglecta 6x (UUXtXtNN), Ae. juvenalis (DcDcXcXcUU), and Ae. vavilovii (DcDcXcXcSsSs) regularly form bivalents at metaphase I. However, in Ae. crassa 6x (DcDcXcXcDD) 0.27 quadrivalents per cell were observed probably as a consequence of the partial homology displayed by the D and Dc genomes. Likewise, the synthetic amphiploid Ae. ventricosa-Secale cereale (DDNNRR) is fertile and displays a diploid-like behavior at metaphase I, despite its recent origin. The pattern of synapsis at late zygotene and pachytene in the natural and artificial allohexaploids was analyzed by whole-mount surface-spreading of synaptonemal complexes under an electron microscope. It revealed that chromosomes were mostly associated as bivalents in all cases, the mean of multivalents per nucleus ranging from 0.17 (Ae. neglecta 6x) to 1.03 (Ae. crassa 6x) in the natural species and 1.05 in the Ae. ventricosa-S. cereale amphiploid. It can be concluded that the mechanism controlling bivalent formation in these species and also in the synthetic amphiploid acts mainly at zygotene by restricting synapsis to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in the homoeologous associations. These observations are discussed in relation to the origin and evolution of the mechanism of diploidization in the allopolyploid species of the Poaceae family.

In plants, small non-coding RNAs (≈20-30 nt) play a major role in a gene regulation mechanism that controls development, maintains heterochromatin and defends against viruses. However, their possible role in somatic and germline cell divisions still remains to be ascertained. ARGONAUTE (AGO) proteins are key players in the different small RNA pathways. Arabidopsis contains ten AGO proteins belonging to three distinct phylogenetic clades based on amino acid sequence, namely: AGO1/AGO5/AGO10, AGO2/AGO3/AGO7, and AGO4/AGO6/AGO8/AGO9. To gain new insights on the role of some AGO proteins, we have focused our attention in AGO2, AGO5 and AGO9 by means of the analysis of plants carrying mutations in the corresponding genes. AGO2 plays a role in the natural cis-antisense (nat-siRNA) pathway and is required for an efficient DNA repair. On the other hand, AGO5, involved in miRNA (microRNA)-directed target cleavage, and AGO9, involved in RNA-directed DNA methylation (RdDM), are highly enriched in germline. On these grounds, we have analyzed the effects of these proteins on the meiotic process and also on DNA repair. It was confirmed that AGO2 is involved in DNA repair, also in ago2-1 the mean cell chiasma frequency in pollen mother cells (PMCs) was increased respect to the wild-type. ago5-4 showed a delay in germination time and a slight decrease in fertility, however the meiotic process and chiasma levels were normal. Meiosis in PMCs of ago9-1 was characterized by a high frequency of chromosome interlocks from pachytene to metaphase I, but chiasma frequency and fertility were normal. Also, genotoxicity assays have confirmed that AGO9 is also involved in somatic DNA repair.

Meiotic recombination plays a critical role in achieving accurate chromosome segregation and increasing genetic diversity. Many studies, mostly in yeast, have provided important insights into the coordination and interplay between the proteins involved in the homologous recombination pathway, especially the recombinase RAD51 and its meiotic paralog DMC1. Here we summarize the current progresses on the function of both recombinases, and the CX3 complex encoded by AtRAD51 paralogs, in the plant model species Arabidopsis thaliana. Similarities and differences respect to the function of these proteins in other organisms are also indicated.

No Crossing Over To ensure the correct division of chromosome during the reduction division of meiosis, homologous chromosomes undergo double-strand breaks that—through crossing over and recombination—link the homologs together (and importantly introduce diversity into the genomes of gametes). But only a minority of these crossovers results in recombination—most are directed into non-crossover pathways. Lorenz et al. (p. 1585 ), working in the yeast Schizosaccharomyces pombe , and Crismani et al. (p. 1588 ), working in the higher plant Arabidopsis thaliana , looked for the factors that limit crossovers and promote non-crossover pathways. The homolog of the human Fanconi anemia complementation group M (FANCM) helicase protein was found to be a major meiotic anti-recombinase, which could drive meiotic recombination intermediates into the non-crossover pathway.

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

Recombination between homologous chromosomes is crucial to ensure their proper segregation during meiosis. This is achieved by regulating the choice of recombination template. In mitotic cells, double-strand break repair with the sister chromatid appears to be preferred, whereas interhomolog recombination is favoured during meiosis. However, in the last year, several studies in yeast have shown the importance of the meiotic recombination between sister chromatids. Although this thinking seems to be new, evidences for sister chromatid exchange during meiosis were obtained more than 50 years ago in non-model organisms. In this mini-review, we comment briefly on the most recent advances in this hot topic and also describe observations which suggest the existence of inter-sister repair during meiotic recombination. For instance, the behaviour of mammalian XY bivalents and that of trivalents in heterozygotes for chromosomal rearrangements are cited as examples. The “rediscovering” of the requirement for the sister template, although it seems to occur at a low frequency, will probably prompt further investigations in organisms other than yeast to understand the complexity of the partner choice during meiosis.

A spreading technique was used to perform a structural analysis of prophase I nuclei in pollen mother cells (PMCs) of wild-type Arabidopsis thaliana. In leptotene, all chromosomes developed fully axial elements before a presynaptic alignment was observed. Pairing and synapsis start in regions close to the telomeres at early zygotene. Interstitial synaptonemal complex (SC) stretches were found to occur at several sites per bivalent at mid zygotene. Within individual bivalents, extensive regions of SC formation often existed at the same time as other extensive regions that were unsynapsed. Also in the same nucleus, one bivalent might have several SC segments, while other bivalents have only a few. The classical bouquet was not so evident as in other plant species. Length measurements of the five pachytene bivalents have allowed the elaboration of a pachytene karyotype. Pachytene chromatin compaction in Arabidopsis was significantly less than that observed in the other species analysed and this is paralleled with a higher recombination rate (centimorgans per megabase).

During first meiotic prophase, homologous chromosomes are normally kept together by both crossovers and synaptonemal complexes (SC). In most eukaryotes, the SC disassembles at diplotene, leaving chromosomes joined by chiasmata. The correct co-orientation of bivalents at metaphase I and the reductional segregation at anaphase I are facilitated by chiasmata and sister-chromatid cohesion. In the absence of meiotic reciprocal recombination, homologs are expected to segregate randomly at anaphase I. Here, we have analyzed the segregation of homologous chromosomes at anaphase I in four meiotic mutants of Arabidopsis thaliana, spo11-1-3, dsy1, mpa1, and asy1, which show a high frequency of univalents at diplotene. The segregation pattern of chromosomes 2, 4, and 5 was different in each mutant. Homologous univalents segregated randomly in spo11-1-3, whereas they did not in dsy1 and mpa1. An intermediate situation was observed in asy1. Also, we have found a parallelism between this behavior and the synaptic pattern displayed by each mutant. Thus, whereas spo11-1-3 and asy1 showed low amounts of SC stretches, dsy1 and mpa1 showed full synapsis. These findings suggest that in Arabidopsis there is a system, depending on the SC formation, that would facilitate regular disjunction of homologous univalents to opposite poles at anaphase I.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation., Author Summary Homologous recombination (HR) during prophase I of meiosis leads to the formation of physical connections, known as chiasmata, between homologous chromosomes (homologs). Chiasmata are essential for accurate homolog segregation at the first meiotic division. HR is initiated by the formation of DNA double-strand breaks (DSBs). As DNA replication prior to meiosis results in the duplication of each homolog to form two identical sister chromatids, a DSB in one sister chromatid could potentially be repaired using the other as the repair template rather than one of the two non-sister chromatids of the homolog. If this route were predominant, the formation of chiasmata would be disfavored and chromosome segregation would be compromised. However, during meiosis there is a strong bias towards inter-homolog recombination (IHR). In this study we have identified AtASY3, a component of the proteinaceous axes that organize the chromosomes during meiosis in Arabidopsis. We find that AtASY3 interacts with AtASY1, a previously identified axis protein that is essential for crossover formation. We show that loss of AtASY3 disrupts the axis-organization of AtASY1. This results in a substantial reduction in chiasmata, and there is extensive chromosome mis-segregation. We propose that loss of AtASY3 affects the efficiency of the inter-homolog bias.